Proton Motive Force-Dependent Hoechst 33342 Transport by the ABC Transporter LmrA of Lactococcus lactis

The fluorescent compound Hoechst 33342 is a substrate for many multidrug resistance (MDR) transporters and is widely used to characterize their transport activity. We have constructed mutants of the adenosine triphosphate (ATP) binding cassette (ABC)-type MDR transporter LmrA of Lactococcus lactis that are defective in ATP hydrolysis. These mutants and wild-type LmrA exhibited an atypical behavior in the Hoechst 33342 transport assay. In membrane vesicles, Hoechst 33342 transport was shown to be independent of the ATPase activity of LmrA, and it was not inhibited by orthovanadate but sensitive to uncouplers that collapse the proton gradient and to N,N'-dicyclohexylcarbodiimide, an inhibitor of the F0F1-ATPase. In contrast, transport of Hoechst 33342 by the homologous, heterodimeric MDR transporter LmrCD showed a normal ATP dependence and was insensitive to uncouplers of the proton gradient. With intact cells, expression of LmrA resulted in an increased rate of Hoechst 33342 influx while LmrCD caused a decrease in the rate of Hoechst 33342 influx. Cellular toxicity assays using a triple knockout strain, i.e., L. lactis ΔlmrA ΔlmrCD, demonstrate that expression of LmrCD protects cells against the growth inhibitory effects of Hoechst 33342, while in the presence of LmrA, cells are more susceptible to Hoechst 33342. Our data demonstrate that the LmrA-mediated Hoechst 33342 transport in membrane vesicles is influenced by the transmembrane pH gradient due to a pH-dependent partitioning of Hoechst 33342 into the membrane.

Effect of homogenised and pasteurised versus native cows' milk on gastrointestinal symptoms, intestinal pressure and postprandial lipid metabolism

Some people experience gastrointestinal symptoms related to cow's milk consumption even if neither lactose intolerance nor cow's milk allergy can be diagnosed. To investigate whether milk homogenization could cause gastrointestinal problems, homogenized and pasteurized milk and native milk were served to eleven volunteers who reported such sensitivity in a random order together with an ingestible pressure measuring capsule. Postprandial lipemia did not differ between the two milk types, but significant differences were found in the postprandial plasma fatty acid composition. No significant difference was found in the amount of gastrointestinal symptoms or in the intestinal pressure after the consumption of native and processed milk. However, the obtained results on pressure in the large intestine (P = 0.068) as well as reported symptoms (P = 0.103) suggest that further studies in this area are needed with a bigger subject group and with longer exposure times to differently processed milk types. (C) 2017 Elsevier Ltd. All rights reserved

Recruitment methods in Alzheimer's disease research: general practice versus population based screening by mail

<p>Abstract</p> <p>Background</p> <p>In Alzheimer's disease (AD) research patients are usually recruited from clinical practice, memory clinics or nursing homes. Lack of standardised inclusion and diagnostic criteria is a major concern in current AD studies. The aim of the study was to explore whether patient characteristics differ between study samples recruited from general practice and from a population based screening by mail within the same geographic areas in rural Northern Norway.</p> <p>Methods</p> <p>An interventional study in nine municipalities with 70000 inhabitants was designed. Patients were recruited from general practice or by population based screening of cognitive function by mail. We sent a questionnaire to 11807 individuals ≥ 65 years of age of whom 3767 responded. Among these, 438 individuals whose answers raised a suspicion of cognitive impairment were invited to an extended cognitive and clinical examination. Descriptive statistics, chi-square, independent sample t-test and analyses of covariance adjusted for possible confounders were used.</p> <p>Results</p> <p>The final study samples included 100 patients recruited by screening and 87 from general practice. Screening through mail recruited younger and more self-reliant male patients with a higher MMSE sum score, whereas older women with more severe cognitive impairment were recruited from general practice. Adjustment for age did not alter the statistically significant differences of cognitive function, self-reliance and gender distribution between patients recruited by screening and from general practice.</p> <p>Conclusions</p> <p>Different recruitment procedures of individuals with cognitive impairment provided study samples with different demographic characteristics. Initial cognitive screening by mail, preceding extended cognitive testing and clinical examination may be a suitable recruitment strategy in studies of early stage AD.</p> <p>Clinical Registration</p> <p>ClinicalTrial.gov Identifier: NCT00443014</p

Long-Term Care Insurance and Carers' Labor Supply: A Structural Model

In Germany, individuals in need of long-term care receive support through benefits of the long-term care insurance. A central goal of the insurance is to support informal care provided by family members. Care recipients can choose between benefits in kind (formal home care services) and benefits in cash. From a budgetary perspective family care is a cost-saving alternative to formal home care and to stationary nursing care. However, the opportunity costs resulting from reduced labor supply of the carer are often overlooked. We focus on the labor supply decision of family carers and the incentives set by the long-term care insurance. We estimate a structural model of labor supply and the choice of benefits of family carers. We find that benefits in kind have small positive effects on labor supply. Labor supply elasticities of cash benefits are larger and negative. If both types of benefits increase, negative labor supply effects are offset to a large extent

Delineating Electrogenic Reactions during Lactose/H+ Symport†

Electrogenic reactions accompanying downhill lactose/H+ symport catalyzed by the lactose permease of Escherichia coli (LacY) have been assessed using solid-supported membrane-based electrophysiology with improved time resolution. Rates of charge translocation generated by purified LacY reconstituted into proteoliposomes were analyzed over a pH range from 5.2 to 8.5, which allows characterization of two electrogenic steps in the transport mechanism: (i) a weak electrogenic reaction triggered by sugar binding and observed under conditions where H+ translocation is abolished either by acidic pH or by a Glu325 -> Ala mutation in the H+ binding site (this step with a rate constant of ~200 s-1 for wildtype LacY leads to an intermediate proposed to represent an “occluded” state) and (ii) a major electrogenic reaction corresponding to 94% of the total charge translocated at pH 8, which is pH-dependent with a maximum rate of ~30 s-1 and a pK of 7.5. This partial reaction is assigned to rate-limiting H+ release on the cytoplasmic side of LacY during turnover. These findings together with previous electrophysiological results and biochemical-biophysical studies are included in an overall kinetic mechanism that allows delineation of the electrogenic steps in the reaction pathway

Polymorphisms in Gag spacer peptide 1 confer varying levels of resistance to the HIV- 1maturation inhibitor bevirimat

Background: The maturation inhibitor bevirimat (BVM) potently inhibits human immunodeficiency virus type 1 (HIV-1) replication by blocking capsid-spacer peptide 1 (CA-SP1) cleavage. Recent clinical trials demonstrated that a significant proportion of HIV-1-infected patients do not respond to BVM. A patient’s failure to respond correlated with baseline polymorphisms at SP1 residues 6-8. Results: In this study, we demonstrate that varying levels of BVM resistance are associated with point mutations at these residues. BVM susceptibility was maintained by SP1-Q6A, -Q6H and -T8A mutations. However, an SP1-V7A mutation conferred high-level BVM resistance and SP1-V7M and T8Δ mutations conferred intermediate levels of BVM resistance. Conclusions: Future exploitation of the CA-SP1 cleavage site as an antiretroviral drug target will need to overcome the baseline variability in the SP1 region of Gag.Publisher PDFPeer reviewe

Range-Finding Risk Assessment of Inhalation Exposure to Nanodiamonds in a Laboratory Environment

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